User:Karmella Haynes/Notebook/BioBrick cloning/2013/04/18: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2013/04/18 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
(7 intermediate revisions by the same user not shown)
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==mm/dd/yy==
==04/18/13==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* MV9 cloning - sequencing order
* Line item 2




----
----
'''Minipreps'''<br>
'''MV9 sequencing'''<br>
* Check with E/P digests
* DNASU
 
** DNA: 200 ng/ 10 μL
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
** Primers: 16 pmol (16 μM) / 10 μL
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
 
----
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
 
* Digests (Fermentas FD)
** Specific notes
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
|-
| DNA (plasmid) || up to 25 μL
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}




Line 70: Line 23:
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. 1-3 || 0.054 || 1.825 || 54.37
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 2. 3-5 || 0.047 || 1.73 || 47.14
|}
 
 
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA (clean digest) || up to 17 μL (500 ng)
|-
| 10x buffer d.p. || 2.0
|-
|-
| phosphatase || 1.0
| 3. 3-6 || 0.131 || 1.87 || 130.79
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}
|}




* Ligations
* Sequencing results for order# 7177
{| {{table}} cellspacing="3" <!-- Ligations table -->
** MV9 1-3/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
|- bgcolor=#cfcfcf
** MV9 3-5/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
** MV9 3-6/ DD122B (fwd): correct sequence but backwards; reads through DD123B (rev primer) on other side
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
| 2. vector(c/d)/ ## ng || &nbsp;
|}


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|-
| Insert DNA        || ###  || ---  ||
|-
| Vector DNA        || ###  || ###  ||
|-
| 2x lgn buf (Roche) || ###  || ###  ||
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
| dH<sub>2</sub>O    || ###  || ###  ||
|-
| &nbsp;            || # μL || # μL ||
|}


----
* Keep MV9 1-3 in vector stocks. See [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/04/12 colony PCR from 04/12/13] for gel confirmation.
'''Oligo annealing'''
* Keep MV9 3-6 in case it might be useful
# New BB 1
# New BB 2


{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
| 10x annealing buffer || 2.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}




Revision as of 12:50, 3 September 2013

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

04/18/13

  • MV9 cloning - sequencing order


MV9 sequencing

  • DNASU
    • DNA: 200 ng/ 10 μL
    • Primers: 16 pmol (16 μM) / 10 μL


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. 1-3 0.054 1.825 54.37
2. 3-5 0.047 1.73 47.14
3. 3-6 0.131 1.87 130.79


  • Sequencing results for order# 7177
    • MV9 1-3/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
    • MV9 3-5/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
    • MV9 3-6/ DD122B (fwd): correct sequence but backwards; reads through DD123B (rev primer) on other side


  • Keep MV9 1-3 in vector stocks. See colony PCR from 04/12/13 for gel confirmation.
  • Keep MV9 3-6 in case it might be useful



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/04/18&oldid=723004"